GENERAL INFORMATION Project Title Characterization of a

GENERAL INFORMATION
Project Title
Characterization of a circulating miRNA signature in response to targeted therapy in Diffuse
Intrinsic Pontine Glioma (DIPG) patients
Institutions/Units involved in the project
1. Functional Genomics and Bioinformatics Department of Experimental Oncology and
Molecular Medicine Fondazione IRCCS Istituto Nazionale dei Tumori - Milano
2. Human Tumors Immunobiology Unit, Department of Experimental Oncology and
Molecular Medicine, IRCCS Istituto Nazionale dei Tumori - Milano
3. Pediatric Unit, Fondazione IRCCS Istituto Nazionale Tumori - Milano
Principal Investigator
Loris De Cecco, Ph.D.
Date of birth: 03.04.1973
Functional Genomics and Bioinformatics
Department of Experimental Oncology and Molecular Medicine
Fondazione IRCCS Istituto Nazionale dei Tumori - Milano
Tel. 02 23905130
e-mail: [email protected]
Project duration
24 months
Budget
The total budget is 121.000 euro
1
Abstract (Inglese)
Hypothesis. The present proposal is a translational research to define biomarkers associated with
response to therapy and with clinical outcome in diffuse intrinsic pontine glioma (DIPG) patients
enrolled in a clinical trial based on radiotherapy, concomitant nimotuzumab and vinorelbine. The
main hypothesis of the project is that levels of specific sets of circulating miRNAs are associated
with response to treatment and/or with clinical outcome. This hypothesis is supported by robust
preliminary data.
Goals. 1. To identify biomarkers by non-invasive and easily accessible methods to improve
personalized DIPG management programs. 2. To achieve independent validation of a miRNA
signature. This is an essential step to confirm the accuracy of a model in predicting the outcome in
DIPG children treated with nimotuzumab/vinorelbine combination. To this end an independent
cohort of patients will be explored. 3. To evaluate miRNA expression in longitudinal time point
series. 4. To investigate the functional role of the miRNA found in goal 1) by in-vitro cellular
models.
Background. DIPG. DIPG, a disease primarily of early school-aged children, is characterized by
rapid onset of symptoms in a previously healthy patient, and by pathognomonic MRI findings.
Diffuse tumors are currently associated with a very poor prognosis due to their unresectable nature
and devastating neurologic symptoms. Radiation is effective as a palliative intervention in a
majority of cases, providing transient symptomatic improvement. Without radiation, median
survival is approximately 4 months. Subsequent tumor progression is almost common, with median
overall survival between 8 and 11 months.
miRNA. miRNAs are single-stranded, non coding RNA molecules of 19-24 nucleotides in length
that are generated by sequential cleavage of primary transcripts (pri-miRNAs) and hairloop
precursors (pre-miRNAs). To date, more than 2000 human miRNAs have been found according to
miRBase and the number of listed miRNAs is increasing. Many studies have so far pointed out that
miRNAs expression is deregulated in several different types of tumors. miRNA expression patterns
are unique in individual tumors and different between cancer and normal tissues. This specificity
and the remarkable stability in a broad range of specimen types (FFPE, plasma and other bodly
fluids) make miRNAs useful biomarkers. Recently, expression analysis of circulating miRNA in
peripheral blood was demonstrated to be altered in cancer patients, proving their clinical relevance
for diagnosis and prognosis.
Clinical significance. At present none biomarker for predicting DIPG prognosis has been entered
into the clinical practice. It is essential to develop better tool able to delineate the biological variants
of gliomas. Without this approach, treatment targets may be missed and patients receiving toxic
therapies not sufficiently targeted to their glioma subtype. Such a biomarker approach means that
patients with the same histological diagnosis, tumor location, and co-morbidities may receive
differing therapy based on the molecular characteristics of their tumors.
Design and methods. On the basis of our previous experience and availability of clinical samples
from an institutional trial, we will assess the expression of circulating miRNA signature predictive
of the treatment and then functional role by genetic manipulation.
2
Abstract (Italiano)
Ipotesi di lavoro. La presente proposta e’ un progetto di ricerca translazionale condotto su
campioni serici da pazienti con glioma pontino intrinseco diffuso (DIPG) arrualota in un trial
clinico basato sul trattamento con radioterapia, nimotuzumab e vinorelbine e che ha per obbettivo
la definizione di biomarcatori associati all’esito clinico del trattamento. L’ipotesi principale del
progetto e’ che i livelli di espressione di specifici miRNA circolanti siano associati alla risposta al
trattamento. Questa ipotesi e’ supportata da robusti dati preliminari.
Scopo della ricerca. Il progetto si prefigge i seguenti scopi: 1) identificare biomarcatori accessibili
con metodi non-invasivi; 2) effettuare una validazione indipendente della firma molecolare
identificata; 3) valutare l’espressione dei miRNA circolanti in una serie longitudinale di campioni
raccolti durante il follow-up dei pazienti; 4) valutare il ruolo funzionale dei miRNA identificati
sfruttando modelli cellulari in-vitro.
Background. DIPG e’ una patologia che colpisce principalmente bambini/ragazzi in eta’ scolare
ed e’ caratterizzata dal rapido emergere dei sintomi legati alla malattia in soggetti precedentemente
sani. Il tumore ha una prognosi estremamente sfavorevole anche in conseguemnza dell’
impossibilita’ di intervenire chirurgicamente. La diagnosi avviene attraverso MRI e il trattamento
attraverso radioterapia e’ attualmente la cura primaria ma questo trattamento risulta nella maggior
parte dei casi in miglioramenti solo transitori. Senza radioterapia la mediana di soppravvivenza e’
circa di 4 mesi mentre con radioterapia la mediana di sopravvivenza si attesta tra 8 e 11 mesi. I
miRNA sono molecole non codificanti di RNA a singolo filamento di 19-24 nucleotidi generate dal
processamento di trascritti primari (pri-miRNAs) e precursori a forma di forcina (pre-miRNAs).
Attualmente sono noti piu’ di 2000 miRNA umani. E’ oramai noto che l’espressione dei miRNA
viene deregolata nei tumori e la possibilita’ di rilevarli in diversi tipi di campioni incluse le biopsie
liquide (plasma, siero, urine, saliva) rende queste molecole estremente interessanti ai fini dell’
identificazione di marcatori biomolecolari. Recentemente l’analisi dell’espressione dei miRNA
circolanti presenti nel sangue si e’ dimostrato alterato in pazienti affetti da diverse patologie
oncologiche.
Implicazioni nella clinica. Attualmente non esistono biomarcatori capaci di predire la prognosi in
pazienti DIPG. E’ essenziale sviluppare migliori strumenti diagnostici/prognostici in quanto la loro
assenza puo’ portare al trattamento inutile di soggetti che invece potrebbero beneficiare di
trattamenti alternativi. Pertanto questo approccio comporta che tumori con la stessa diagnosi,
localizzazione e comorbidita’ possano essere indirizzati verso trattamenti spefici diversi basati sulle
caratteristiche molecolari del tumore.
Disegno dello studio e metodi. Sulla base della esperienza come messo in evidenza nei risultati
prelimiari e della disponibilita’ di campioni da un trial, valuteremo l’espressione dei miRNA
circolanti e il loro ruolo funzionale.
3
Background and rationale
Diffuse intrinsic pontine glioma (DIPG), a disease primarily of early school-aged children, is
characterized by rapid onset of symptoms in a previously healthy patient, and by pathognomonic
MRI findings. Without radiation, median survival is approximately 4 months (Lassman , 1967).
Radiation is effective as a palliative intervention in a majority of cases, providing transient
symptomatic improvement. Subsequent tumor progression is almost universal, with median overall
survival between 8 and 11 months, and overall survival of approximately 30% at 1 year and less
than 10% at 2 years (Hargrave , 2006; Bradley, 2013). Rare (2–3%) long-term survival has been
reported, usually associated with atypical imaging and clinical features at presentation (Pollack et
al, 2011; Jackson et al, 2013). Diffuse tumors constitute the majority (75–80%) of brain tumors and
are associated with a very poor prognosis due to their unresectable nature, devastating neurologic
symptoms associated with the local extension, and poor response to adjuvant therapy. The median
time to tumor progression is 5–6 months, with a median survival of 9–12 months despite treatment
(Freeman et al, 1998).
At present none biomarker for predicting prognosis has been entered into the clinical practice. It is
essential to develop better tools able to delineate the biological variants of gliomas. Without this
approach, treatment targets may be missed and patients given toxic therapies not sufficiently
targeted to their glioma subtype. Such a biomarker approach means that patients with the same
histological diagnosis, tumor location, and co-morbidities may receive differing therapy based on
the molecular characteristics of their tumors.
miRNAs are single-stranded, non coding RNA molecules of 19-24 nucleotides in length that are
generated by sequential cleavage of primary transcripts (pri-miRNAs) and hairloop precursors (premiRNAs)(Kusenda, 2006). To date, more than 2000 human miRNAs have been found according to
miRBase and the number of listed miRNAs is increasing (miRBase: www.miBase.org). Many
studies have so far pointed out that miRNAs expression is deregulated in several different types of
tumors (Bardel, 2011). miRNA expression patterns are unique in individuals tumors and different
between cancer and normal tissues (Volinia, 2006). This specificity and the remarkable stability in a
broad range of specimen types (FFPE, plasma and other body fluids) make miRNAs useful
biomarkers (Cortez, 2011). Recently expression analysis of circulating miRNA in peripheral blood
was demonstrated to be altered in cancer patients, proving their clinical relevance for diagnosis and
prognosis (Zhu, 2009). The relationship between circulating miRNAs and cancer tissue was first
investigated by Mitchell et al (Mitchell, 2008) showing that circulating miRNAs originate from
cancer tissues. This study, supported by others, demonstrate that the level of circulating miRNAs
reflects the miRNome of tumor tissue, providing evidence on the hypothesis that circulating
miRNAs can serve as ideal biomarkers. Further studies elucidated how circulating miRNAs are
actively secreted into the blood stream. Increasing proofs support the idea that circulating miRNA
are incorporated into exosomes or are bound to RNA-binding proteins, explaining their stability
against RNA digestion (Valadi, 2007).
Currently a variety of miRNA detection methods including northern blotting, in situ hybridization,
qRT-PCR, microarrays and deep sequencing are available. The procedures for detection of
circulating miRNAs are the same as that for cellular miRNAs, except for the extremely low amount
of starting material. High-throughput profiling techniques such as microarray and miRNA deep
sequencing are effective tools to obtain expression profiles. Since these methods generally required
a relative large volume of blood, they are used for the initial discovery screening. Then for
validation approaches, qRT-PCR methodologies have proved to be useful in detecting the low level
of circulating miRNA expression.
4
Preliminary results
With the aim of exploring add-on strategies, a non-randomized, open label phase II pilot study was
conducted at Fondazione IRCCS Istituto Nazionale dei Tumori (Milan) to assess the efficacy in
terms of objective response rate according to the RECIST criteria of combining nimotuzumab and
vinorelbine with radiation in newly-diagnosed DIPG. Vinorelbine was administered at 20 mg/m2
weekly together with nimotuzumab at 150 mg/m2 during the first 12 weeks of treatment, when
radiotherapy was delivered from week
10 miRNA predictive signature
Kaplan-Meier curves for risk groups obtained from 10CV:
3 to 9 at total dose of 54 Gy.
Vinorelbine at 25 mg/m2 was given
p=4.27E-05; HR= 4.33 95%CI 1.49-12.54
any other week, with the same dose of
nimotuzumab, thereafter until tumor
progression or for a total of two years.
Twenty-five children (median age
7.4) were enrolled according to the
standard MRI inclusion criteria and a
follow-up with a median observation
time of 21 months was achieved. A
response was observed in 24/25
patients (96 %). One-year PFS rates
was 30 ± 10 % respectively; the
median PFS were 8.5 months.
Serum specimens were collected at
baseline and during follow-up. We
used a high-throughput microRNA screening approach to assess miRNA profile in serum samples
obtained from 24 DIPG patients with the main aim to identify novel non-invasive biomarkers able
to improve the prediction of disease outcome and therapeutic sensitivity. Here we present the
preliminary results of serum miRNA profiling at baseline. microRNA expression profiling was
performed using Agilent platform and Human miRNA SureSelect 8x60K containing 2006 miRNAs
annotated on miRBase19.0. Primary data analysis yielded a matrix containing 330 detectable
miRNA. Association with PFS allowed us to disclose a signature of 10 miRNAs able to stratify
high and low risk patients (HR=4.33, 95%CI 1.49-12.54; p=4.27E-05).
The 10-miRNA signature was validated by RTqPCR and its accuracy in predicting the response to
treatment in DIPG patients was confirmed. At present, this is the first study that aims at
investigating circulating miRNA levels in DIPG pediatric patients.
HIGH
Two patients were
RISK
selected for evaluation
of the 10 miRNA
LOW
MiRNA ID
expression
in
RISK
10
8
longitudinal analysis
1
2
(up to 30 serum
3
6
9
samples
for
each
7
4
patient, collected from
5
diagnosis till relapse or
last
control)
and
associated to clinical
data.
Pt 1
Pt 4
Primers were obtained
from Exiqon (Vedbæk,
Denmark). qRT-PCR
Relapse
was performed using
Relapse
5
the miRCURY LNA™ Universal RT microRNA PCR system (Exiqon) and following the
manufacturer’s instructions. Real-time was run using QuantStudio 12flex. Normalization was
performed using has-miR16 and 3 invariant miRNAs identified in the microarray profiling. Results
are reported as –ΔCt. Shaded orange area identifies the antibody/vinorelbine treatment. Shaded blue
area identifies the radio therapy Black bar identifies relapse. The preliminary results were promising
since different patterns of miRNA expression were recorded between low and high risk patients
suggesting that the relative expression of the signature could potentially predict disease outcome.
Study aims.
In order to evaluate the accuracy of the miRNA signature to foresee treatment outcome in DIPG
patients, the following activities are required:
A) independent validation of our miRNA signature. This is an essential step to confirm the
accuracy of our model in predicting the outcome in DIPG children treated with
nimotuzumab/vinorelbine combination. An independent cohort of patients is required to this
aim. We will use the serum specimens collected in the frame of “phase II open label
randomized study of radiotherapy, concomitant nimotuzumab and vinorelbine and reirradiation at relapse versus radiotherapy and multiple elective radiotherapy courses with
concomitant nimotuzumab and vinorelbine for newly diagnosed childhood and adolescence
DIPG” (PI Dr. M. Massimino).
B) Evaluation of predictive power of our miRNA signature. Our signature will be challenged
against a cohort of children not affected by neurological pathologies or affected by
neurological pathologies but not treated with nimotuzumab and vinorelbine match by age
and gender. This is an essential point to ascertain the accuracy of our signature to predict the
response to nimotuzumab and vinorelbine treatment in DIPG patients. In alternative we will
assess the accuracy of our miRNA model to be a prognostic tool of neurological pathologies
in children.
C) Evaluation of miRNA expression in longitudinal time point series. First of all, we will
extend the number of cases under evaluation. Based on the expression during time we will
monitor the efficacy of treatment and the ability to predict the time of relapse.
D) Functional role of miRNA. Most of the miRNA present in our signature were only recently
annotated in miRBase (miRBase_v19.0; August 2013). Really few information is available
on their biological functions. For this reason we propose to investigate their functional role
exploiting in vitro functional models.
Expected Results
The promising utility of liquid biopsies has recently gained great attention toward its clinical
application in the management of cancer patients. As matter of fact, to improve the clinical outcome
of DIPG patients, accurate detection and monitoring of disease are necessary. At present, surgical
and/or biopsy specimens are generally used to detect tumor alterations and to build treatment
predictive models. However, the collection of this kind of material due to its invasive nature is not
feasible in DIPG children. In addition, the tumor at baseline may fail to reflect the current tumor
dynamics and drug sensitivity that may change during the therapeutic process. Therefore, liquid
biopsies monitoring the presence of cell free miRNAs could be the ideal material for developing
non-invasive biomarkers helping to define personalized DIPG management programs.
6
Personnel involved into the project
NAME
Time for the
project (%)
50
Biologist, PhD, he has wide experience in high-throughput
methodologies. He will take care of project coordination. He
will oversee the experimental activities and will contribute to the
computational analyses, predictor design/refinement. He will
coordinate the UNIT-A of the project.
30
Biologist, responsible of Functional Genomics core facility, will
oversee and coordinate the activities and the genomic aspects.
She will participate on the activities of UNIT-A.
30
MD, Oncologist, director of the pediatric unit. She will
contribute to the planning project. She is responsible of the
“phase II open label randomized study of radiotherapy,
concomitant nimotuzumab and vinorelbine and re-irradiation at
relapse versus radiotherapy and multiple elective radiotherapy
courses with concomitant nimotuzumab and vinorelbine for
newly diagnosed childhood and adolescence DIPG”. She will
coordinate the UNIT-B of the project.
30
Biologist, head Human Tumors Immunobiology Unit, he will
oversee the functional genomics analyses. He will coordinate
the UNIT-C of the project.
100
He has experience in molecular biology and in high-throughput
methodologies and he will perform the experimental activities
needed in the project. He will participate on the activities of
UNIT-A.
100
Experience in molecular biology and cellular biology
methodologies. He/she will perform the functional activities
needed for the project. He/She will participate on the activities
of UNIT-B.
Loris De Cecco
(03/04/73)
Role on project
PI
Silvana Canevari
(30/11/1948)
Internal
collaborator
Maura Massimino
(8/03/1962)
Internal
collaborator
Andrea Anichini
( 19/02/1952)
Internal
collaborator
Marco
Giannoccaro
(01/09/1986)
Fellow
TBD
()
Fellow
The principal investigator and all other personnel of the research team have already successfully
collaborated. Evidence lines in the recent paper on nimotuzumab and vinorelbine treatment of DIPG
7
patients (Massimino et al. Results of nimotuzumab and vinorelbine, radiation and re-irradiation for
diffuse pontine glioma in childhood. J Neurooncol. 2014 Jun;118:305-12). In addition the results
were presented at the EACR-AACR-SIC conference (Florence , 20-23 June 2015).
BRIEF CURRICULUM: Maura Massimino, MD
Maura Massimino was born on March 8, 1962.
She graduated in Milan University in 1987 with laude. She achieved Hematology Board in 1991
and Pediatrics Board in 1995 at Milan University. From 1987 onwards she worked at the Pediatric
Oncology Unit of Istituto Nazionale Tumori of Milan, first as resident until 1989, afterwards as
permanent staff member, from May 2009 as Deputy Director and from July 2010 as Director. Her
clinical research is particularly devoted to childhood brain tumors, lymphomas and thyroid
cancer.Co-chair for the SIOP brain tumor subcommittee of the Ependymoma study group since
2007, chair since 2012. She is also involved in late effects evaluation both from the
endocrinological and neuro-cognitive point of views.She is the author of more than 200 published
full papers, more than 350 meeting communications and several book chapters. She is married,
mother of two children.
BRIEF CURRICULUM: Silvana Canevari, PhD
Born on November 30, 1948
Graduated in Milan University in 1972 with laude, PhD in Cancer Immunology in 1974. From 1973
onwards she worked at the Experimental Department of Fondazione IRCCS Istituto Nazionale
Tumori of Milan, first as fellow until 1974, afterwards as permanent staff member. From 2008 to
2013 she was Director of the Molecular Therapies Unit and afterward she works as associate
researcher to Scientific Direction for the facility of Functional Genomics and Bioinformatics. Her
experimental research was devoted initially to the immunological characterization and subsequently
to the molecular characterization of human tumors. Since 2007 she is responsible of The Functional
Genomics and Bioinformatics facility.She is the author of more than 200 published full papers,
more than 350 meeting communications and several book chapters concerning different aspects of
the experimental oncology, immunology, biochemistry and biotechnology applied to human tumors.
BRIEF CURRICULUM: Andrea Anichini, PhD
Born on November February, 2nd , 1952
Dr. Anichini is member of the Italian Society of Cancerology and of EACR. He has published 147
papers on peer reviewed journals an has an H index=37.He acts as reviewer for international
science journals, including Science Transl. Med., J. Exp. Med., Cancer Res. and other journals. He
has been awarded grants from AIRC and from italian and european funding agencies since 1992. He
is mentoring four fellows. He has devoted most of the past three decades to research in tumor
immunobiology with emphasis on melanoma and B cell lymphomas. He has contributed to
identification of anti‐tumor HLA‐restricted T cells in melanoma patients, identification of
melanocyte lineage antigens as immunogenic epitopes, understanding of development of antitumor
immunity during progression, molecular identification of tumor antigens and mechanism of
8
response to anti‐CTLA‐4. Dr. Andrea Anichini is author of 147 peer-reviewed publications and 24
book chapters concerning different aspects of the biology and immune response to human tumors.
BRIEF CURRICULUM: Marco Giannoccaro
Born on November September, 1st , 1986
Dr. Giannoccaro, as member of Functional Genomics Unit at the Fondazione IRCCS National
Cancer Institute, is responsible for the extraction of nucleic acids from frozen tissue, FFPE and
biofluids. In addition he is responsible of quality control through the use of RTqPCR
QuantStudio12K-flex platform in order to ensure adequate quality for microarray analysis. He has
good experience in processing samples for microarray analysis (labeling, purification), miRNA and
gene-expression profiling by Agilent and Illumina platforms and gene-expression DASLwg
tecnology for FFPE samples.
BRIEF CURRICULUM: TBD
Degree in biology or biotechnology. Young researcher with experience in microarray, highthroughput methodologies, including data analysis, molecular biology, and cellular biology
methodologies.
PERT chart (Program Evaluation Review Technique)
UNIT-A
Circulating miRNA
Identification and
quantification
METHODS
The present proposal is a translational research linked to “phase II open label randomized study of
radiotherapy, concomitant nimotuzumab and vinorelbine and re-irradiation at relapse versus
radiotherapy and multiple elective radiotherapy courses with concomitant nimotuzumab and
vinorelbine for newly diagnosed childhood and adolescence DIPG”. Collection of blood specimens
will be carried out at baseline (before treatment) and during follow-up.
Here below the main features of the trial are summarized
Inclusion criteria
9
1.
Patients from 2 to 21 years old will be eligible;
2.
No previous treatment consented apart from steroids;
3.
Strict eligibility criteria will radiologically-verified DIPG (an intrinsic, pontine-based
infiltrative lesion hypointense on T1- and hyperintense on T2-weighted sequences, involving at
least 2/3 of the pons) (Hargrave et al, 2006);
4.
symptoms lasting less than 6 months, life expectancy ≥ 4 weeks; Karnowski/Lansky
performance status ≥40%;
5.
no organ dysfunction; no pregnancy or breast-feeding;
6.
Patients undergo baseline cranial MRI with gadolinium, to be repeated if treatment begins
more than 2 weeks; spinal MRI due to the occurrence of metastatic cases at diagnosis will also be
mandatory;
7.
Written and signed informed consent from parents or legal guardians will be obtained before
starting the treatment.
Treatment design
Pre-therapy examinations (within 14 days prior to the start of therapy):
•
Cranial and holospinal MRI with gadolinium (estimation of index lesion);
•
Full clinical examination including neurological, body weight, height, Karnowski or Lansky
performance status;
•
Full blood count, creatinine, electrolytes, Ca, Mg, phosphate, AST, LDH, total protein,
albumin, CRP, bilirubin, blood sugar; coagulation tests (Quick value or INR, PTT, TT);
•
Pregnancy test in females of childbearing age (must be negative).
•
Evaluation of quality of life and life situation.
Standard arm: medical treatment
Nimotuzumab + Vinorelbine and full-dose radiotherapy: standard arm
Nimotuzumab 150 mg /m²/d as iv short-term infusion for 30 min weekly in week 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12 and Vinorelbine 20 mg/m²/d weekly in week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12 as iv short-term infusion for 30 min (Induction phase);
1st re-evaluation week 12 (day 84-92). In case of non-progressive disease: Nimotuzumab
150 mg/m²/d iv short-term infusion for 30 min and Vinorelbine 25 mg/m²/d as iv short-term
infusion for 30 min biweekly in week 14, 16, 18, 20, 22, 24 (Consolidation phase I);
2nd re-evaluation week 24, thereafter in case of non-progressive disease: Nimotuzumab 150
mg/m²/d iv short-term infusion for 30 min and Vinorelbine 25 mg/m²/d as iv short-term infusion for
30 min biweekly, with reevaluation at week 36 until progression or maximum at week 104
10
Experimental arm
Pre-therapy examinations like in standard arm.
Nimotuzumab + Vinorelbine and refracted radiotherapy doses: experimental arm
Nimotuzumab 150 mg/m²/d as iv short-term infusion for 30 min weekly in week 1, 2, 3, 4, 5,
6,7, 8,9,10, 11, 12 and Vinorelbine weekly 20 mg/m²/d in week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 as
iv short-term infusion for 30 min (Induction phase, as for standard arm);
1st re-evaluation week 12. In case of non-progressive disease, any other week,
Nimotuzumab 150 mg/m2 as iv short-term infusion for 30 min and Vinorelbine 25 mg/m²/d as iv
short-term infusion for 30 min for a total duration of 104 weeks;
2nd re-evaluation week 24, thereafter in case of non-progressive disease re-irradiation one
for a total of 19.8 Gy in 11 fractions at 1.8 Gy/day from week 25 to week 27 together with
vinorelbine/nimotuzumab continuation any other week;
3rd re-evaluation week 36, thereafter in case of non-progressive disease
vinorelbine/nimotuzumab continuation any other week;
4th re-evaluation week 44, thereafter in case of non-progressive disease: re-irradiation two
for a total of 19.8 Gy in 11 fractions at 1.8 Gy/day from week 45 to week 47 together with
vinorelbine/nimotuzumab continuation any other week;
Further re-evaluation will be done at week 60 and thereafter any 12 weeks as for standard
arm continuing vinorelbine and nimotuzumab until progression or until full duration of treatment
for 104 weeks. Patients will continue with re-irradiation courses also in case of progressive disease,
and will continue to be evaluated for OS. The intended sample size is N = 54 patients. A stratified
randomization procedure will be used to randomize eligible, consenting patients to experimental or
control arms (1:1 ratio). Randomization will occur using a computer generated randomized number
sequence. Stratification factors, demonstrated to be significantly associated with patient outcome in
a previous study (Massimino 2014) will be patients age (≤4 years age, >4 years) and need for
shunting at diagnosis (no, yes).
Collection of serum specimens.
Serum provides the liquid portion of the blood without cells and clotting factors and, therefore,
should contain proteins and other molecules that represent the whole body system. The cells and
clotting factors must be removed from the blood sample by allowing adequate time for a clot to
form. Most manufacturers of collections systems for serum samples recommend 30–60 min at room
temperature for a clot to form and longer if the subject was taking any kind of anticoagulant at
sample collection
Blood from initial diagnosis, from specific time points during treatment, and during follow-up, will
be collected. The blood sample are/will be processed within 1 h after collection by centrifugation at
1,300 x g at 4°C for 20 min, and plasma separated in aliquots in fresh tubes and stored at -80°C.
11
The samples will be sent to the Functional and Bioinformatics (INT) for RNA extraction and
profiling.
miRNA profiling
The miRNA microarray experiments will be performed by the Functional Genomics of the
Department of Experimental Oncology and Molecular Medicine (Fondazione IRCCS Istituto
Tumori) using Human miRNA SurePrint Microarray (Agilent Technology). Since the number of
miRNAs is still increasing with the everyday discovery of new miRNAs, microarrays are at present
one the most used methodology for assessing the expression of miRNAs in tissue samples, enabling
the cheap and reliable profiling on the last available miRBase version. For each miRNA, multiple
probes are spotted on the array. The average intensity of these probes will be calculated and used as
expression value of the miRNA for further analyses. For each plasma sample, total RNA will be
extracted, labeled, hybridized with the miRNA array and further processed following the
manufacturer's protocols. The arrays will be scanned using an Agilent Technology G2565CA
scanner and the scanned images will be processed using the Feature Extraction software package
version 10.5 (Agilent Technology) in order to extract the raw data. Validation in an independent
cohort of samples of our 10 miRNA signature will be carried out. Moreover we will test the miRNA
expression in autopsy material that has undergone only a small amount of postmortem degradation.
Data analysis
Analysis will be performed using BrB-Array Tools v4.2.1 stable release developed by Dr. Richard
Simon (NCI) and BRB-Array Tools development team (EMMES corporation). miRNAs expression
will also be related to clinical outcome; the analyses will be performed separately for each selected
miRNA. Time will be calculated as the interval between patient randomization and disease
progression or death, whichever will occur first, with censoring at the date of last follow up control
for patients alive and without documented progression. We will use a multivariable Cox
proportional hazard model in which miRNA expression will be evaluated as continuous variable;
the clinical and histological parameters will be included in the model as adjustment factors. The
performance of the model including the miRNA under evaluation in terms of discriminative ability
will be evaluated by calculating the bootstrap corrected Harrell C statistic and compared with that
of the model not including the miRNA, with the aim of evaluating whether the expression data
provides more accuracy than that associated to clinical/histological parameters only.
In vitro models
As neoplastic tissues from these patients are not available, the possibility of carrying out functional
studies with DIPG cell lines would strongly improve the quality/meaning of the results that we are
obtaining by analysis of serum samples. To this end, we plan exploit DIPG cell lines to:
i)
assess the miRNA content in exosomes derived from the cell lines.
12
ii)
silence/overexpress miRNA of interest in cell lines for functional characterization. To this
end, we will analyze changes in gene expression (by whole genome expression analysis), survival,
and proliferation (by apoptosis/proliferation assays) of the cell lines where miRNA of interest have
been silenced or overexpressed.
Timetable
UNIT
Aim
A) Independent validation
Activities
1 ° year
2 ° year
RNA extraction; quality check
and microarray analysis of the
entire case material
Bioinformatics analysis of
microarray data
Statistical analysis
Validation of microarray data by
qRT-PCR
UNIT-A
B) Evaluation of predictive
power of our signature
Collection of sera specimens
Microarray analysis of validation
set
Bioinformatics analysis of
microarray data
Statistical analysis
Integration of genomic and
clinical data
RNA extraction
qRT-PCR analysis of our 10
C) Evaluation of miRNA miRNA signature
Bioinformatics analysis of
expression in
microarray data
longitudinal series
Statistical analysis to develop
model associated to type and time
of treatment
selection of patients
UNIT-B
D) Clinical trial
treatment
longitudinal blood withdrawal
patients' follow-up
UNIT-C
exosome extraction form blood
and cell cultures
D) Functional role of selected genetic manipulation of cell
culturs: knock-in
miRNAs
genetic manipulation of cell
culturs: knock-out
References
-Bardel DP, microRNAs: genomics, biogenesis, mechanism, and function. Cell 116 (2): 281-297
(2004)
-Bradley KA, Zhou T, McNall-Knapp RY, Jakacki RI, Levy AS,Vezina G, Pollack IF. Motexafingadolinium and involved field radiation therapy for intrinsic pontine glioma of childhood: a
children’s oncology group phase 2 study. Int J Radiat Oncol Biol Phys (2013); 85:e55–e60
-Cortez MA, Bueso-Ramos C, Ferdin J et al microRNAs in body fluids – the mix of hormones and
biomarkers. Nat Rev Clin Oncol 8: 467-477 (2011)
-Freeman CR, Farmer JP. Pediatric brain stem gliomas: a review. Int J Radiat Phys. (1998); 40:265–
71
-Hargrave D, Bartels U, Bouffet E. Diffuse brainstem glioma in children: critical review of clinical
trials. Lancet Oncol. (2006); 7:241–248
13
-Kusenda B, Mraz M, Mayer J et al. microRNA biogenesis, functionality and cancer relevance.
Biomed Pap 150: 205-215 (2006)
-Lassman LP, Arjona VE. Pontine gliomas of childhood. Lancet (1967); 1:913–915
-Massimino M, Biassoni V, Miceli R, Schiavello E, Warmuth-Metz M, Modena P, Casanova M,
Pecori E, Giangaspero F, Antonelli M, Buttarelli FR, Potepan P, Pollo B, Nunziata R, Spreafico F,
Podda M, Anichini A, Clerici CA, Sardi I, De Cecco L, Bode U, Bach F, Gandola L. Results of
nimotuzumab and vinorelbine, radiation and re-irradiation for diffuse pontine glioma in childhood. J
Neurooncol. (2014); 118(2):305-312
-Mitchell PS, Parkin RK, Kroh EM et al Circulating microRNAs as stable blood-based markers for
cancer detection Proc Natl Acad Sci USA 105: 10513-10518 (2008)
-Valadi H, Ekstrom K, Bossios A, et al Exosome-mediated transfer of mRNAs and miRNAs is a
novel mechanism of genetic exchange between cells. Nat Cell Biol 9: 654-659 (2007)
-Volinia S A microRNA expression signature of human solid tumors defines cancer gene targets.
Proc Natl Acad Sci USA 103: 2257-2261 (2006)
-Zhu W, Qin W, Atasoy U et al. Circulating microRNAs in breast cancer and healthy subjects.
BMC Res Notes 2: 89 (2009)
Budget
Personell costs
Consumables
Meeting and travel costs
Other (publication costs)
subtotal total cost/year
Overheads (adminstration office)10%
1st year 2nd year Total
38,000
38,000
76,000
15,000
15,000
30,000
1,000
1,000
2,000
1,000
1,000
2,000
55,000
55,000
subtotal
110,000
5,500
5,500
Total
cost
121,000
i. Personnel costs: Two fellowships; 38.000,00 Euro/year (tot 76.000,00 euro)
ii. Supplies/consumables: miRNA microarray chips and kits: 5.000 euro/year; miRNA extraction
kits: 2,000 euro/year; RTqPCR assays and reagents: 4,000 euro/year; reagents for cell culture: 4,000
euro/year (tot. 30.000,00 euro)
iii. Equipment: none to be imputed to this grant
iv. Patient care costs: none
14
v. Other expenses (descriptive line item list):
1,000 euro/year for travelling and meetings (tot. 2.000,00 euro)
1.000,00 euro/year for publication costs (tot. 2.000,00 euro)
Overheads (administration office)10% 5,500/year (tot. 11,000 euro)
Total requested: 121,000 euro
Co-found:
NOTE: the activities of UNIT-B including the “phase II open label randomized study of
radiotherapy, concomitant nimotuzumab and vinorelbine and re-irradiation at relapse versus
radiotherapy and multiple elective radiotherapy courses with concomitant nimotuzumab and
vinorelbine for newly diagnosed childhood and adolescence DIPG” are covered by other funds
(see attachment).
Principal Investigator CV
Name
Loris De Cecco
Place and date of birth
Citizenship
Lab address
Milan, Italy
E-mail
Chene-Bougeries (CH), 03/04/1973
Italian
Fondazione IRCCS Istituto Nazionale dei Tumori Via Antonio Amadeo 42,20133
[email protected]
Education:
• 2009 PhD in Life and Biomolecular Sciences, Open University (London, UK)
• 1998 Degree in Biological Sciences, University of Trieste.
Career path:
• 2007 – present
Member of the Functional Genomics Unit, Fondazione IRCCS Istituto
Nazionale Tumori, Milan (directed by Dr. S. Canevari)
15
• 2004 – 2007 PhD programme (Open University) under supervision of Dr. M. Pierotti. Istituto
Nazionale Tumori, Milan
• 1999 – 2003 FIRC and AIRC fellowship, Molecular genetics of cancer unit, IFOM (Fondazione
FIRC di Oncologia Molecolare), Milan. Director: Dr M.Pierotti
Since 2000 Dr. De Cecco’s scientific activity has been focused on the analysis of gene expression
patterns underlying the progression and the clinical outcome in different types of tumors. He has an
extensive experience and background in molecular biology techniques and microarray procedures,
including microarray manufacturing, synthesis of fluorescent probes, and hybridization. Since 2007
as member of the Functional Genomics Unit at Fondazione IRCCS Istituto Nazionale Tumori, he
has carried out microarray studies using the Illumina and Agilent platforms and has a deep
knowledge in protocols for gene-expression, for miRNA profiling and methylation analysis. In
addition he has a long experience in handling and processing microarray data, including excellent
skills in the use of microarray tools for data analysis (BrB-Arraytools, MeV, R Biocondactor) and
for pathway identification (DAVID, GeneSpring GX, Partek Genomic Suite, Ingenuity Pathway
Analysis, CytoScape). He has experience in whole-exome NGS analysis with SOLiD
(LifeTechnologies) platform, including library construction and sequencing. He is co-author of
more than 40 scientific papers.
Awards: gold medal as young scientist for his contribution on cancer research by gene-expression
analysis using high-throughput cDNA microarray platforms (2006; Associazione Amici di Milano).
Grants: “Rule of IL2-family members in CLL” founded by Compagnia di S.Paolo Foundation
(Genoa) (2012; 10.000 euro).
Role in the project: Dr. De Cecco will oversee and coordinate all the activities, he will be
responsible for study design and for the planning and development of the research; he will oversee
the bioinformatics analysis required for the project and he will maintain the relationships with all
collaborators. As PI, he will be full dedicated to the project and he will be last author of the
publications related to the results of the project.
Papers indexed in PubMed (2010-2015):
In the last 5 years, the PI is first author of 8 papers as first author and 1 paper as last author. Total
I.F. =158
1:Triulzi T*, De Cecco L*, Sandri M, Prat A, Giussani M, Paolini B, Carcangiu ML, Canevari S,
Bottini A, Balsari A, Menard S, Generali D, Campiglio M, Di Cosimo S, Tagliabue E. Wholetranscriptome analysis links trastuzumab sensitivity of breast tumors to both HER2 dependence and
immune cell infiltration. Oncotarget. 2015 Jul 1.
2: De Cecco L, Capaia M, Zupo S, Cutrona G, Matis S, Brizzolara A, Orengo AM, Croce M,
Marchesi E, Ferrarini M, Canevari S, Ferrini S. Interleukin 21 Controls mRNA and MicroRNA
16
Expression in CD40-Activated Chronic Lymphocytic Leukemia Cells. PLoS One. 2015 Aug
25;10(8):e0134706.
3: Vitali C, Bassani C, Chiodoni C, Fellini E, Guarnotta C, Miotti S, Sangaletti S, Fuligni F, De
Cecco L, Piccaluga PP, Colombo MP, Tripodo C. SOCS2 controls proliferation and stemness of
hematopoietic cells under stress conditions and its deregulation marks unfavourable acute
leukemias. Cancer Res. 2015 Apr 9.
4: De Cecco L, Nicolau M, Giannoccaro M, Daidone MG, Bossi P, Locati L, Licitra L, Canevari S.
Head and neck cancer subtypes with biological and clinical relevance: Meta-analysis of geneexpression data. Oncotarget. 2015 Mar 20.
5: Huang X, Dugo M, Callari M, Sandri M, De Cecco L, Valeri B, Carcangiu ML, Xue J, Bi R,
Veneroni S, Daidone MG, Ménard S, Tagliabue E, Shao Z, Wu J, Orlandi R. Molecular portrait of
breast cancer in China reveals comprehensive transcriptomic likeness to Caucasian breast cancer
and low prevalence of luminal A subtype. Cancer Med. 2015 Mar 18.
5: De Cecco L*, Negri T*, Brich S, Mauro V, Bozzi F, Dagrada G, Disciglio V, Sanfilippo R,
Gronchi A, D'Incalci M, Casali PG, Canevari S, Pierotti MA, Pilotti S. Identification of a gene
expression driven progression pathway in myxoid liposarcoma. Oncotarget. 2014 Aug
15;5(15):5965-77.
6: De Cecco L, Bossi P, Locati L, Canevari S, Licitra L. Comprehensive gene expression metaanalysis of head and neck squamous cell carcinoma microarray data defines a robust survival
predictor. Ann Oncol. 2014 Aug;25(8):1628-35.
7: Massimino M, Biassoni V, Miceli R, Schiavello E, Warmuth-Metz M, Modena P, Casanova M,
Pecori E, Giangaspero F, Antonelli M, Buttarelli FR, Potepan P, Pollo B, Nunziata R, Spreafico F,
Podda M, Anichini A, Clerici CA, Sardi I, De Cecco L, Bode U, Bach F, Gandola L. Results of
nimotuzumab and vinorelbine, radiation and re-irradiation for diffuse pontine glioma in childhood. J
Neurooncol. 2014 Jun;118(2):305-12. doi: 10.1007/s11060-014-1428-z. Epub 2014 Apr 3.
8: Minna E, Romeo P, De Cecco L, Dugo M, Cassinelli G, Pilotti S, Degl'Innocenti D, Lanzi C,
Casalini P, Pierotti MA, Greco A, Borrello MG. miR-199a-3p displays tumor suppressor functions
in papillary thyroid carcinoma. Oncotarget. 2014 May 15;5(9):2513-28.
9: Dassano A, Colombo F, Trincucci G, Frullanti E, Galvan A, Pettinicchio A, De Cecco L,
Borrego A, Martinez Ibañez OC, Dragani TA, Manenti G. Mouse pulmonary adenoma
susceptibility 1 locus is an expression QTL modulating Kras-4A. PloS Genet. 2014 Apr
17;10(4):e1004307.
10: Granata A, Nicoletti R, Tinaglia V, De Cecco L, Pisanu ME, Ricci A, Podo F, Canevari S, Iorio
E, Bagnoli M, Mezzanzanica D. Choline kinase-alpha by regulating cell aggressiveness and drug
sensitivity is a potential druggable target for ovarian cancer. Br J Cancer. 2014 Jan 21;110(2):33040.
11: Vallacchi V, Vergani E, Camisaschi C, Deho P, Cabras AD, Sensi M, De Cecco L, Bassani N,
Ambrogi F, Carbone A, Crippa F, Vergani B, Frati P, Arienti F, Patuzzo R, Villa A, Biganzoli E,
17
Canevari S, Santinami M, Castelli C, Rivoltini L, Rodolfo M. Transcriptional profiling of
melanoma sentinel nodes identify patients with poor outcome and reveal an association of CD30(+)
T lymphocytes with progression. Cancer Res. 2014 Jan 1;74(1):130-40.
12: Crippa E, Lusa L, De Cecco L, Marchesi E, Calin GA, Radice P, Manoukian S, Peissel B,
Daidone MG, Gariboldi M, Pierotti MA. miR-342 regulates BRCA1 expression through modulation
of ID4 in breast cancer. PLoS One. 2014 Jan 27;9(1):e87039.
13: Dassano A, Mancuso M, Giardullo P, De Cecco L, Ciuffreda P, Santaniello E, Saran A,
Dragani TA, Colombo F. N(6)-isopentenyladenosine and analogs activate the NRF2-mediated
antioxidant response. Redox Biol. 2014 Mar 6;2:580-9.
14: Galvan A, Frullanti E, Anderlini M, Manenti G, Noci S, Dugo M, Ambrogi F, De Cecco L,
Spinelli R, Piazza R, Pirola A, Gambacorti-Passerini C, Incarbone M, Alloisio M, Tosi D, Nosotti
M, Santambrogio L, Pastorino U, Dragani TA. Gene expression signature of non-involved lung
tissue associated with survival in lung adenocarcinoma patients. Carcinogenesis. 2013
Dec;34(12):2767-73.
15: Callari M, Tiberio P, De Cecco L, Cavadini E, Dugo M, Ghimenti C, Daidone MG, Canevari S,
Appierto V. Feasibility of circulating miRNA microarray analysis from archival plasma samples.
Anal Biochem. 2013 Jun 15;437(2):123-5.
16: De Cecco L, Dugo M, Canevari S, Daidone MG, Callari M. Measuring microRNA expression
levels in oncology: from samples to data analysis. Crit Rev Oncog. 2013;18(4):273-87. Review.
17: Tiberio P, De Cecco L, Callari M, Cavadini E, Daidone MG, Appierto V. MicroRNA detection
in plasma samples: how to treat heparinized plasma. J Mol Diagn. 2013 Jan;15(1):138-9.
18: De Cecco L, Berardi M, Sommariva M, Cataldo A, Canevari S, Mezzanzanica D, Iorio MV,
Tagliabue E, Balsari A. Increased sensitivity to chemotherapy induced by CpG-ODN treatment is
mediated by microRNA modulation. PLoS One. 2013;8(3):e58849.
19: Musella V, Verderio P, Reid JF, Pizzamiglio S, Gariboldi M, Callari M, Milione M, De Cecco
L, Veneroni S, Pierotti MA, Daidone MG. Effects of warm ischemic time on gene expression
profiling in colorectal cancer tissues and normal mucosa. PLoS One. 2013;8(1)
20: Frullanti E, Colombo F, Falvella FS, Galvan A, Noci S, De Cecco L, Incarbone M, Alloisio M,
Santambrogio L, Nosotti M, Tosi D, Pastorino U, Dragani TA. Association of lung adenocarcinoma
clinical stage with gene expression pattern in noninvolved lung tissue. Int J Cancer. 2012 Sep
1;131(5)
21: Rumio C, Sommariva M, Sfondrini L, Palazzo M, Morelli D, Viganò L, De Cecco L, Tagliabue
E, Balsari A. Induction of Paneth cell degranulation by orally administered Toll-like receptor
ligands. J Cell Physiol. 2012 Mar;227(3):1107-13.
22: Sommariva M, De Cecco L, Tagliabue E, Balsari A. Modulation of DNA repair genes induced
by TLR9 agonists: A strategy to eliminate "altered" cells? Oncoimmunology. 2012 Mar 1;1(2):258259.
18
23: Callari M, Dugo M, Musella V, Marchesi E, Chiorino G, Grand MM, Pierotti MA, Daidone
MG, Canevari S, De Cecco L. Comparison of microarray platforms for measuring differential
microRNA expression in paired normal/cancer colon tissues. PLoS One. 2012;7(9)
24: Bagnoli M*, De Cecco L*, Granata A, Nicoletti R, Marchesi E, Alberti P, Valeri B, Libra M,
Barbareschi M, Raspagliesi F, Mezzanzanica D, Canevari S. Identification of a chrXq27.3
microRNA cluster associated with early relapse in advanced stage ovarian cancer patients.
Oncotarget. 2011 Dec;2(12):1265-78. * equally contributing first authors
25: Sommariva M, De Cecco L, De Cesare M, Sfondrini L, Ménard S, Melani C, Delia D,
Zaffaroni N, Pratesi G, Uva V, Tagliabue E, Balsari A. TLR9 agonists oppositely modulate DNA
repair genes in tumor versus immune cells and enhance chemotherapy effects. Cancer Res. 2011
Oct 15;71(20):6382-90.
26: Callari M, Cappelletti V, De Cecco L, Musella V, Miodini P, Veneroni S, Gariboldi M, Pierotti
MA, Daidone MG. Gene expression analysis reveals a different transcriptomic landscape in female
and male breast cancer. Breast Cancer Res Treat. 2011 Jun;127(3):601-10.
27: Mezzanzanica D, Canevari S, Cecco LD, Bagnoli M. miRNA control of apoptotic programs:
focus on ovarian cancer. Expert Rev Mol Diagn. 2011 Apr;11(3):277-86. doi: 10.1586/erm.11.1.
Review.
28: Fernández-Ramires R, Gómez G, Muñoz-Repeto I, de Cecco L, Llort G, Cazorla A, Blanco I,
Gariboldi M, Pierotti MA, Benítez J, Osorio A. Transcriptional characteristics of familial nonBRCA1/BRCA2 breast tumors. Int J Cancer. 2011 Jun 1;128(11):2635-44.
29: Mezzanzanica D, Bagnoli M, De Cecco L, Valeri B, Canevari S. Role of microRNAs in ovarian
cancer pathogenesis and potential clinical implications. Int J Biochem Cell Biol. 2010
Aug;42(8):1262-72.
30: Franco MD, Colombo F, Galvan A, Cecco LD, Spada E, Milani S, Ibanez OM, Dragani TA.
Transcriptome of normal lung distinguishes mouse lines with different susceptibility to
inflammation and to lung tumorigenesis. Cancer Lett. 2010 Aug 28;294(2):187-94.
31: Bortolomai I, Canevari S, Facetti I, De Cecco L, Castellano G, Zacchetti A, Alison MR, Miotti
S. Tumor initiating cells: development and critical characterization of a model derived from the
A431 carcinoma cell line forming spheres in suspension. Cell Cycle. 2010 Mar 15;9(6):1194-206.
32: Busacca S, Germano S, De Cecco L, Rinaldi M, Comoglio F, Favero F, Murer B, Mutti L,
Pierotti M, Gaudino G. MicroRNA signature of malignant mesothelioma with potential diagnostic
and prognostic implications. Am J Respir Cell Mol Biol. 2010 Mar;42(3):312-9.
19